ANTIBODY-TUMOR NECROSIS FACTOR alpha FUSION PROTEIN AND ITS PREPARATION AND APPLICATIONS
20220098262 · 2022-03-31
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
C07K16/2863
CHEMISTRY; METALLURGY
C07K2319/90
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07K2317/14
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
A61K31/45
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
A61K38/191
HUMAN NECESSITIES
A61K31/704
HUMAN NECESSITIES
A61K31/704
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
A61K38/191
HUMAN NECESSITIES
International classification
A61K31/45
HUMAN NECESSITIES
A61K31/704
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C07K16/28
CHEMISTRY; METALLURGY
Abstract
Provided is an antibody-tumor necrosis factor α fusion protein and its preparation and applications. Specifically, the present disclosure relates to a fusion protein comprising an antibody moiety and a TNFα moiety, a nucleic acid molecule encoding same, a nucleic acid construct, a host cell and a method of production thereof, as well as applications of these materials in prevention and/or treatment of tumors.
Claims
1. A fusion protein of an antibody and TNFα, comprising an antibody moiety and a TNFα moiety conjugated to the C-terminal of the heavy chain of said antibody, whereby said fusion protein comprises a heavy chain comprising the heavy chain of said antibody with said TNFα moiety linked at the C-terminal optionally through a peptide linker and optionally a signal peptide at N-terminal, and a light chain comprising the light chain of said antibody and optionally a signal peptide at N-terminal; Wherein, said antibody is specific to a tumor antigen; and wherein, said TNFα moiety is selected from the group consisting of TNFα and a functionally equivalent analog or derivative thereof.
2. The fusion protein according to claim 1, further characterized in one or more features selected from the followings: (a) Wherein, the tumor antigen is selected from the group consisting of members of the ErbB family, such as EGFR, Her-2, Her-3 and Her-4; (b) Wherein, said antibody is selected from the group consisting of Trastuzumab, Pertuzumab, T-DM1, Cetuximab, ABX-EGF, and functionally equivalent analogs or derivatives thereof; (c) Wherein, said antibody moiety is in the form of a full-length antibody; (d) Wherein, the heavy chain of said antibody comprises terminal modification to facilitate fusion, such as deletion of one or more lysine residues at the C-terminal; (e) Wherein, said TNFα is selected from the group consisting of human or mammalian TNFα; mature TNFα, secreted TNFα, and transmembrane TNFα; (f) Wherein, said linker peptide is absent or has a length of 1-50, 5-50, 5-40, 10-40 or 10-30 amino acid residues, or a length of an integer from 1 to 50 amino acid residues; and/or (g) Wherein, the molar ratio of said antibody moiety to said TNFα moiety is 1:2.
3. The fusion protein according to claim 1, wherein the antibody is a Her-2 antibody or an EGFR antibody, such as Trastuzumab, Cetuximab or a functionally equivalent analog or derivative thereof; and optionally, the TNFα moiety is human TNFα, for example a TNFα molecule having the sequence of positions 469-625 in SEQ ID NO: 2; wherein, for example, the heavy chain of said fusion protein has the amino acid sequence of positions 20-625 or the amino acid sequence of positions 1-625 in SEQ ID NO: 2, and the light chain of said fusion protein has the amino acid sequence of positions 21-234 or the amino acid sequence of positions 1-234 in SEQ ID NO: 4; or the heavy chain of said fusion protein has the amino acid sequence of positions 17-621 or the amino acid sequence of positions 1-621 in SEQ ID NO: 20, and the light chain of said fusion protein has the amino acid sequence of positions 17-230 or the amino acid sequence of positions 1-230 in SEQ ID NO: 22.
4. A nucleic acid construct, comprising or consisting of a nucleic acid molecule encoding the heavy chain of the fusion protein according to claim 1 and/or a complement of said nucleic acid molecule, or comprising or consisting of a nucleic acid molecule selected from the following: A nucleic acid molecule encoding the amino acid sequence of positions 20-625 or positions 1-625 in SEQ ID NO: 2, and/or a complement thereof; or A nucleic acid molecule comprising the nucleotide sequence of positions 58-1875 or positions 1-1875 in SEQ ID NO: 1 and optionally a stop codon, and/or a complement thereof; or A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, and/or a complement thereof; or A nucleic acid molecule encoding the amino acid sequence of positions 17-621 or positions 1-621 in SEQ ID NO: 20, and/or a complement thereof; or A nucleic acid molecule comprising the nucleotide sequence of positions 49-1863 or positions 1-1863 in SEQ ID NO: 19 and optionally a stop codon, and/or a complement thereof; or A nucleic acid molecule having as the nucleotide sequence of SEQ ID NO: 19, and/or a complement thereof.
5. A nucleic acid construct or a combination of nucleic acid constructs, comprising or consisting of a nucleic acid molecule encoding the heavy chain of the fusion protein according to claim 1 and a nucleic acid molecule encoding the light chain of said fusion protein, and/or complements of said nucleic acid molecules; wherein the nucleic acid molecule encoding said heavy chain and/or the complement thereof and the nucleic acid molecule encoding said light chain and/or the complement thereof may be included in the same nucleic acid construct or in two or more nucleic acid constructs; wherein, for example, said nucleic acid construct or combination of nucleic acid constructs comprise: A nucleic acid molecule encoding the amino acid sequence of positions 20-625 or positions 1-625 in SEQ ID NO: 2, and/or the complement thereof, and a nucleic acid molecule encoding the amino acid sequence of positions 21-234 or positions 1-234 in SEQ ID NO: 4, and/or the complement thereof; or A nucleic acid molecule encoding the amino acid sequence of positions 17-621 or positions 1-621 in SEQ ID NO: 20, and/or the complement thereof, and a nucleic acid molecule encoding the amino acid sequence of positions 17-230 or positions 1-230 in SEQ ID NO: 22, and/or the complement thereof; or A nucleic acid molecule comprising the nucleotide sequence of positions 58-1875 or positions 1-1875 in SEQ ID NO: 1 and optionally a stop codon, and/or the complement thereof, and a nucleic acid molecule comprising the nucleotide sequence of positions 61-702 or positions 1-702 in SEQ ID NO: 3 and optionally a stop codon, and/or the complement thereof; or A nucleic acid molecule comprising the nucleotide sequence of positions 49-1863 or positions 1-1863 in SEQ ID NO: 19 and optionally a stop codon, and/or the complement thereof, and a nucleic acid molecule comprising the nucleotide sequence of positions 49-690 or positions 1-690 in SEQ ID NO: 21 and optionally a stop codon, and/or the complement thereof; or A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, and/or the complement thereof, and a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 3, and/or the complement thereof A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 19, and/or the complement thereof, and a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 21, and/or the complement thereof.
6. The nucleic acid construct according to claim 4, wherein said nucleic acid construct comprises or consists of a vector comprising the nucleic acid molecule(s), such as an expression vector.
7-10. (canceled)
11. The nucleic acid construct or the combination of nucleic acid constructs according to claim 5, wherein said nucleic acid construct comprises or consists of a vector comprising said nucleic encoding molecule(s), such as an expression vector.
12. A host cell comprising the nucleic acid construct according to claim 4; wherein, for example, said host cell is selected from the group consisting of bacteria, fungi, yeasts, plant cells and animal cells; wherein, for example, said host cell is a CHO cell.
13. A host cell comprising the nucleic acid construct or combination of nucleic acid constructs according to claim 5; wherein, for example, said host cell is selected from the group consisting of bacteria, fungi, yeasts, plant cells and animal cells; wherein, for example, said host cell is a CHO cell.
14. A method for producing the fusion protein, including: culturing the host cell according to claim 13 under a condition suitable for expression of said fusion protein to express said fusion protein; and isolating the fusion protein; and optionally one or more of the following steps: subjecting culture of the host cell to a stress to obtain a strain having a high expression of the fusion protein, such as culturing under the stress of methotrexate, preferably an increasing gradient of methotrexate stress; and separating and/or purifying the fusion protein.
15. A composition comprising (i) the fusion protein according to claim 1 and, optionally, (ii) one or more additional active agents for treating or for preventing tumors; Wherein, for example, said one or more additional active agents for treating or preventing tumors is(are) selected from chemotherapeutic agents and radiotherapeutic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, botanical anticancer medicines, hormones and immunotherapeutics; specifically, mitosis inhibitors, transcription inhibitors and protein synthesis inhibitors; and more specifically, vinblastine, vincristine, vindesine, vinorelbine, colchicamine, colchicine, colchicamide, podophyllotoxin, etoposide, teniposide, paclitaxel or docetaxel, camptothecin, homoharringtonine, procarbazine, asparaginase, cisplatin, carboplatin, mitoxantrone, tamoxifen, cyclophosphamide, chlorambucil hydrochloride, lomustine, semustine, cetepa, busulfan, methamphetamine, chlorambucil, fluorouracil, Tegafur, efluidin, carmofur, mercaptopurine, methotrexate, cytarabine, cyclocytidine, mercaptoguanine, hexamethylmelamine, hydroxyurea, mitomycin, doxorubicin, epirubicin, bleomycin, pelomycin, atorvastatin, gleevec, gemcitabine, actinomycin D, cycloheximide, topotecan and/or leuprolide; especially actinomycin D, cycloheximide, doxorubicin; and analogs, derivatives, prodrugs or metabolites thereof.
16. A composition comprising (i) the nucleic acid construct or the combination of nucleic acid constructs according to claim 5 and, optionally, (ii) one or more additional active agents for treating or for preventing tumors; Wherein, for example, said one or more additional active agents for treating or preventing tumors is(are) selected from chemotherapeutic agents and radiotherapeutic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, botanical anticancer medicines, hormones and immunotherapeutics; specifically, mitosis inhibitors, transcription inhibitors and protein synthesis inhibitors; and more specifically, vinblastine, vincristine, vindesine, vinorelbine, colchicamine, colchicine, colchicamide, podophyllotoxin, etoposide, teniposide, paclitaxel or docetaxel, camptothecin, homoharringtonine, procarbazine, asparaginase, cisplatin, carboplatin, mitoxantrone, tamoxifen, cyclophosphamide, chlorambucil hydrochloride, lomustine, semustine, cetepa, busulfan, methamphetamine, chlorambucil, fluorouracil, Tegafur, efluidin, carmofur, mercaptopurine, methotrexate, cytarabine, cyclocytidine, mercaptoguanine, hexamethylmelamine, hydroxyurea, mitomycin, doxorubicin, epirubicin, bleomycin, pelomycin, atorvastatin, gleevec, gemcitabine, actinomycin D, cycloheximide, topotecan and/or leuprolide; especially actinomycin D, cycloheximide, doxorubicin; and analogs, derivatives, prodrugs or metabolites thereof.
17. A composition comprising (i) the host cell according to claim 13; and, optionally (ii) one or more additional active agents for treating or for preventing tumors; Wherein, for example, said one or more additional active agents for treating or preventing tumors is(are) selected from chemotherapeutic agents and radiotherapeutic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, botanical anticancer medicines, hormones and immunotherapeutics; specifically, mitosis inhibitors, transcription inhibitors and protein synthesis inhibitors; and more specifically, vinblastine, vincristine, vindesine, vinorelbine, colchicamine, colchicine, colchicamide, podophyllotoxin, etoposide, teniposide, paclitaxel or docetaxel, camptothecin, homoharringtonine, procarbazine, asparaginase, cisplatin, carboplatin, mitoxantrone, tamoxifen, cyclophosphamide, chlorambucil hydrochloride, lomustine, semustine, cetepa, busulfan, methamphetamine, chlorambucil, fluorouracil, Tegafur, efluidin, carmofur, mercaptopurine, methotrexate, cytarabine, cyclocytidine, mercaptoguanine, hexamethylmelamine, hydroxyurea, mitomycin, doxorubicin, epirubicin, bleomycin, pelomycin, atorvastatin, gleevec, gemcitabine, actinomycin D, cycloheximide, topotecan and/or leuprolide; especially actinomycin D, cycloheximide, doxorubicin; and analogs, derivatives, prodrugs or metabolites thereof.
18. A method for inducing apoptosis in tumor cells, inhibiting growth of tumor and/or treating a tumor, wherein said method comprises administering to a subject in need thereof a therapeutically effective amount of the fusion protein according to claim 1; wherein, said tumor is selected from tumors having high expression of a member of the ErbB family such as EGFR, Her-2, Her-3, Her-4 and tumors that are responsive to TNFα treatment, such as gastric cancer, breast cancer, ovarian cancer, melanoma, bowel cancer such as colon and rectal cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, head and neck cancer, cervical cancer, skin cancer, esophageal cancer, bone cancer and leukemia.
19. A method for inducing apoptosis in tumor cells, inhibiting growth of tumor and/or treating a tumor, wherein said method comprises administering to a subject in need thereof a therapeutically effective amount of the nucleic acid construct or a combination of nucleic acid constructs according to claim 5; wherein, said tumor is selected from tumors having high expression of a member of the ErbB family such as EGFR, Her-2, Her-3, Her-4 and tumors that are responsive to TNFα treatment, such as gastric cancer, breast cancer, ovarian cancer, melanoma, bowel cancer such as colon and rectal cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, head and neck cancer, cervical cancer, skin cancer, esophageal cancer, bone cancer and leukemia.
20. A method for inducing apoptosis in tumor cells, inhibiting growth of tumor and/or treating a tumor, wherein said method comprises administering to a subject in need thereof a therapeutically effective amount of the host cell according to claim 13; wherein, said tumor is selected from tumors having high expression of a member of the ErbB family such as EGFR, Her-2, Her-3, Her-4 and tumors that are responsive to TNFα treatment, such as gastric cancer, breast cancer, ovarian cancer, melanoma, bowel cancer such as colon and rectal cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, head and neck cancer, cervical cancer, skin cancer, esophageal cancer, bone cancer and leukemia.
21. A method for inducing apoptosis in tumor cells, inhibiting growth of tumor and/or treating a tumor, wherein said method comprises administering to a subject in need thereof a therapeutically effective amount of composition according to claim 15; wherein, said tumor is selected from tumors having high expression of a member of the ErbB family such as EGFR, Her-2, Her-3, Her-4 and tumors that are responsive to TNFα treatment, such as gastric cancer, breast cancer, ovarian cancer, melanoma, bowel cancer such as colon and rectal cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, head and neck cancer, cervical cancer, skin cancer, esophageal cancer, bone cancer and leukemia.
22. A method for inducing apoptosis in tumor cells, inhibiting growth of tumor and/or treating a tumor, wherein said method comprises administering to a subject in need thereof a therapeutically effective amount of composition according to claim 16; wherein, said tumor is selected from tumors having high expression of a member of the ErbB family such as EGFR, Her-2, Her-3, Her-4 and tumors that are responsive to TNFα treatment, such as gastric cancer, breast cancer, ovarian cancer, melanoma, bowel cancer such as colon and rectal cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, head and neck cancer, cervical cancer, skin cancer, esophageal cancer, bone cancer and leukemia.
23. A method for inducing apoptosis in tumor cells, inhibiting growth of tumor and/or treating a tumor, wherein said method comprises administering to a subject in need thereof a therapeutically effective amount of composition according to claim 17; wherein, said tumor is selected from tumors having high expression of a member of the ErbB family such as EGFR, Her-2, Her-3, Her-4 and tumors that are responsive to TNFα treatment, such as gastric cancer, breast cancer, ovarian cancer, melanoma, bowel cancer such as colon and rectal cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, head and neck cancer, cervical cancer, skin cancer, esophageal cancer, bone cancer and leukemia.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The drawings are provided for purpose of illustration and exemplification, with no effect of limiting the scope of the invention.
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DETAILED DESCRIPTION OF THE INVENTION
[0034] In the following, the present invention will be described in details with reference to specific embodiments.
[0035] This application is based on the finding that TNFα can be conjugated to an antibody molecule to provide a dual-target antibody-TNFα fusion protein which exhibits strong tumor-specific effects including inhibition, killing and apoptosis-induction in vitro and in vivo. Furthermore, these fusion proteins in combination with an additional anti-tumor active, such as a chemotherapeutic agent, provide an effect of synergism in killing, inhibition and apoptosis-induction in tumor cells in vitro and in vivo.
[0036] The inventors designed and produced an antibody-TNFα fusion protein with multiple biological activities. Specifically, the inventors, using the technology of molecular cloning, constructed an antibody-TNFα fusion protein by conjugating an antibody against a tumor-specific molecule on cell surface or a molecule over-expressed by tumor cells (e.g. tumor-specific antigens and tumor-associated antigens) with tumor necrosis factor α (TNFα) which is capable of tumor-killing and activation of anti-tumor immunity. The antibody moiety recruits the fusion protein to the environment around the tumor cells, producing actions on the tumor cells including inhibition, killing and induced apoptosis. The fusion protein carries the TNFα molecule as conjugated with the antibody to the site of tumor, where the TNFα molecule forms a transmembrane format with the tumor-specific molecule on surface and kills the tumor cells. Further, in presence of an active agent that is cytotoxic to tumor (e.g., a chemotherapeutic agent), the antibody-TNFα fusion protein of the invention and the active agent provide an enhancement of synergism in killing tumor cells.
[0037] The invention has overcome various technical obstacles, including difficulties in recombinant protein expression, difficulties in obtaining a productive strain with high expression of the product, and the risk that the expressed recombinant protein fails to fold into the correct configuration and is thus insoluble or prone to aggregate or biologically inactive, and succeeded in a recombinant protein molecule that can be effectively expressed in mammalian cells.
[0038] The fusion protein of the invention is stable in expression, highly biologically active and is effective in treating and preventing tumors. For example, the fusion protein of the invention can effectively inhibit osteogenic and cartilage differentiation, and can be effectively used for treating cancers, such as gastric cancer, pancreatic cancer, breast cancer, ovarian cancer, melanoma and colon cancer. The fusion protein of the invention can also be used in combination with an additional anti-tumor agent to provide a synergistic anti-tumor effect.
[0039] As used herein, the open-ended terms such as “comprise”, “have” and “include” and their grammatical variants encompass the scenarios as meant by the closed-ended terms such as “consist of . . . ”, “composed of . . . ” and “is . . . ”. In the present application, a numerical range constitutes a disclosure including the values at two ends, every integer and fractions between the ends, and all ranges formed between values of said ends, integers and fractions. For multiple ranges described for a single parameter, ends of these ranges can be recombined the new ranges obtained thereby are also deemed as specifically disclosed herein. In the present application, the features and elements can be recombined across embodiments and examples and the embodiments and examples obtained thereby are also deemed as specifically disclosed herein.
[0040] Fusion Protein and its Components
[0041] The fusion proteins of the invention is a fusion protein between an antibody and TNFα, which is also referred to herein below as “antibody-TNFα fusion protein” or “antibody-TNFα” or “the fusion protein of the invention”, as well as synonyms in context. The fusion protein of the invention comprises an antibody moiety and a TNFα moiety conjugated to the C-terminal of the heavy chain of said antibody, wherein the heavy chain of the antibody and the TNFα moiety are fused to form the heavy chain of the fusion protein, and the light chain of the antibody constitutes the light chain of the fusion protein. The heavy chain and the light chain of the fusion protein each may independently and optionally further comprise a signal peptide at N-terminal.
[0042] In a preferred embodiment, the antibody moiety in the fusion protein of the invention is in the form of a full-length antibody. The form of a full-length antibody refers to the complete structure of an immunoglobulin (Ig), except for optional C-terminal modification to facilitate fusion. In particular, the structure of an immunoglobulin refers to the structure of a monomeric Ig molecule such as IgG. In context of the present invention, immunoglobulins include IgG, IgD, IgE, IgA, IgM and sub-classifications and subunits thereof. For example, according to the present invention, the antibody may be an IgG antibody, including those of IgG1, IgG2, IgG3, and IgG4 sub-classes. In a preferred embodiment, as shown in
[0043] In the fusion protein of the invention, the antibody moiety provides specificity to a tumor antigen. Examples of tumor antigen include, but are not limited to, members of the ErbB family, such as EGFR, Her-2, Her-3, and Her-4. Correspondingly, the antibodies include EGFR antibodies, Her-2 antibodies, Her-3 antibodies and Her-4 antibodies. Specifically, examples of antibody include, but are not limited to Trastuzumab, Pertuzumab (Perjeta), T-DM1, Cetuximab, ABX-EGF, and functionally equivalent analogs or derivatives thereof.
[0044] As used herein, the term “functionally equivalent analog or derivative” should be understood in a broad sense as referring to an agent that is highly similar to a specified biomolecule as reference (such as a cellular element, a nucleic acid molecule, a cytokine (e.g., TNFα)) in structure and/or composition or that comprises the backbone structure of the reference and has no significant difference in biological activity as compared with the reference. For example, in the context of nucleic acid molecules and proteins such as antibodies, functionally equivalent analogs or derivatives thereof include molecules that are different from the reference molecule at one or more positions along the nucleotide sequence or the amino acid sequence via mutation of deletion, insertions and/or substitution while having a sequence identity of at least 60%, 70%, 80%, 90%, 95%, 97%, 98%, or even at least 99% to the reference. For example, in the context of TNFα, the functionally equivalent analogs or derivatives thereof have substantially the same or equivalent effect in killing cells and inducing apoptosis.
[0045] In some embodiments, the antibody is a Her-2 antibody or an EGFR antibody. In some embodiments, the antibody is a Her-2 antibody, such as Trastuzumab or a functionally equivalent analog or derivative thereof. In some embodiments, the antibody is Trastuzumab.
[0046] In some embodiments, the antibody is an EGFR antibody, such as Cetuximab or a functional analog or derivative thereof. In some embodiments, the antibody is Cetuximab.
[0047] In some embodiments, the antibody molecule comprises C-terminal modification to facilitate fusion, such as deletion of one or more lysine residues at C-terminal of the heavy chain. For example, the antibody moiety derived from Trastuzumab or Cetuximab may comprise the lysine deletion at C-terminal.
[0048] In the fusion protein of the invention, the TNFα moiety refers to monomeric molecule(s) of TNFα or a functionally equivalent analog or derivative thereof. In some embodiments, the TNFα molecule is selected from: human or mammalian TNFα; mature TNFα, secreted TNFα, and transmembrane TNFα. In some embodiments, the TNFα moiety is a human TNFα. For example, in a fusion protein of the invention, the TNFα moiety has the amino acid sequence of positions 469-625 in SEQ ID NO: 2.
[0049] In a preferred embodiment, the TNFα moiety is directly conjugated to the C-terminal of the heavy chain of the antibody, without a peptide linker. Meanwhile, the TNFα portion can also be conjugated to the heavy chain of the antibody via a peptide linker. The peptide linker, if present, may have a length of 1-50, 5-50, 5-40, 10-40 or 10-30 amino acid residues, or a length of an integer from 1 to 50. A person of skills in the art knows how to determine the presence/absence and length of the peptide linker with conventional analysis and means, see for example PNAS, 95: 5929-5934, 1998; Protein Eng., 13 (5): 309-312, 2000; Protein Eng., 15 (11): 871-879, 2003; etc.
[0050] In a preferred embodiment, the antibody moiety is in the form of the tetramer of a monomeric Ig molecule, and the molar ratio of antibody to TNFα in the form of monomeric molecule(s) is 1:1 or preferably 1:2, as shown in
[0051] In some embodiments, the fusion protein further comprises a signal peptide at N-terminal. As understood, a signal peptide refers to an amino acid sequence that functions to direct secretion, localization and/or transportation of proteins, which usually have a length of 5-30 amino acid residues. Generally, a mature protein harvested in recombinant production does not contain a signal peptide.
[0052] In an exemplary embodiment, the heavy chain of the fusion protein according to the present invention has the amino acid sequence of positions 20-625 (without signal peptide) or positions 1-625 (with signal peptide) in SEQ ID NO: 2, and the light chain of the fusion protein has the amino acid sequence of positions 21-234 (without signal peptide) or positions 1-234 (with signal peptide) in SEQ ID NO: 4. Along the amino acid sequence as set forth by SEQ ID NO: 2, the segment of amino acids (aa) at positions 1-19 is a transmembrane signal peptide, the segment of aa 20-468 is the heavy chain of Trastuzumab with the lysine-deletion at C-terminal, the segment of aa 469-625 is the amino acid sequence of human TNFα. Along the amino acid sequence as set forth by SEQ ID NO: 4, the segment of aa 1-20 is the transmembrane signal peptide, and the segment of aa 21-234 is the light chain of Trastuzumab. SEQ ID NO: 1 is an example of nucleotide sequence encoding the amino acid of SEQ ID NO: 2, wherein the last TGA is the stop codon. SEQ ID NO: 3 is an example of nucleotide sequence encoding SEQ ID NO: 4, wherein the last TGA is the stop codon.
[0053] In another exemplary embodiment, the heavy chain of the fusion protein according to the present invention has the amino acid sequence of positions 17-621 (without signal peptide) or positions 1-621 (with signal peptide) in SEQ ID NO: 20, and the light chain of the fusion protein has the amino acid sequence of positions 17-230 (without signal peptide) or positions 1-230 (with signal peptide) in SEQ ID NO: 22. Along the amino acid sequence as set forth by SEQ ID NO: 20, the segment of amino acids (aa) at positions 1-16 is the transmembrane signal peptide, the segment of aa 17-464 is the heavy chain of Cetuximab with the lysine-deletion at C-terminal, the segment of aa 165-621 is the amino acid sequence of human TNFα. Along the amino acid sequence as set forth by SEQ ID NO: 21, the segment of aa 1-16 is the transmembrane signal peptide, and the segment of aa 17-230 is the light chain of Cetuximab. SEQ ID NO: 19 is an example of nucleotide sequence encoding the amino acid of SEQ ID NO: 20, wherein the last TGA is the stop codon. SEQ ID NO: 21 is an example of nucleotide sequence encoding SEQ ID NO: 22, wherein the last TGA is the stop codon.
[0054] As shown by the examples in the present application, the fusion protein of the invention has one or more activities selected from the group consisting of binding to a tumor antigen, promoting apoptosis in fibroblasts, inducing apoptosis in tumor cells, inhibiting growth of tumor, and providing a synergistic anti-tumor effect in combination with an additional anti-tumor agent.
[0055] The Fusion Protein-Encoding Nucleic Acid Molecules, Nucleic Acid Constructs, Vectors and Host Cells
[0056] As used herein, the term “nucleic acid construct” is chemically synonymous with “nucleic acid molecule”, which is artificial in origin and is not a natural product. Therefore, a nucleic acid construct, as in the case of a nucleic acid molecule, may include another encoding nucleic acid molecule, i.e., a coding sequence, as an integral component thereof, such as is the case of a vector containing a coding sequence. As used herein, the terms “nucleic acid molecule”, “nucleic acid” and “nucleotide sequence” in relation to an encoding sequence or a specified nucleotide sequence are interchangeably synonymous, all encompass the nucleic acid molecule or sequence that is complementary to a specified nucleic acid molecule or sequence.
[0057] Provided herein is a nucleic acid construct, comprising a nucleotide sequence encoding the heavy chain of the fusion protein of the invention, such as a vector comprising said nucleotide sequence. Or, the nucleic acid construct is a nucleic acid molecule encoding the heavy chain of the fusion protein of the invention.
[0058] In an embodiment, a nucleic acid construct according to the invention may be a nucleic acid molecule encoding the fusion protein heavy chain consisting of Trastuzumab heavy chain-human TNFα, or a construct such as a vector comprising said encoding nucleic acid molecule. For example, the nucleic acid construct may comprise or consist of a nucleic acid molecule selected from the followings:
[0059] A nucleic acid molecule encoding the amino acid sequence of aa 20-625 or aa 1-625 in SEQ ID NO: 2; or
[0060] A nucleic acid molecule comprising the nucleotide sequence of positions 58-1875 or positions 1-1875 in SEQ ID NO: 1 and optionally a stop codon; or
[0061] A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1.
[0062] In another embodiment, a nucleic acid construct of the present invention may be a nucleic acid molecule encoding the fusion protein heavy chain consisting of Cetuximab heavy chain-human TNFα, or a construct such as a vector comprising said encoding nucleic acid molecule. Specifically, the nucleic acid construct may comprise or consist of a nucleic acid molecule selected from the followings:
[0063] A nucleic acid molecule encoding the amino acid sequence of positions 17-621 or positions 1-621 in SEQ ID NO: 20; or
[0064] A nucleic acid molecule comprising the nucleotide sequence of positions 49-1863 or positions 1-1863 in SEQ ID NO: 19 and optionally a stop codon; or
[0065] A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 19.
[0066] Also provided herein is a nucleic acid construct or a combination of nucleic acid constructs, comprising or consisting of a nucleic acid molecule encoding the heavy chain of the fusion protein of the invention and a nucleic acid molecule encoding the light chain of the fusion protein of the invention. The nucleic acid molecule encoding the heavy chain of the fusion protein and the nucleic acid molecule encoding the light chain of the fusion protein may be included in the same nucleic acid construct (e.g., a vector) or in two or more nucleic acid constructs (e.g., vectors).
[0067] For example, in some embodiments, when the fusion protein is Trastuzumab-TNFα, correspondingly, said nucleic acid construct or combination of nucleic acid constructs may comprise or consist of the following nucleic acid molecules:
[0068] A nucleic acid molecule encoding the amino acid sequence of positions 20-625 or positions 1-625 in SEQ ID NO: 2, and a nucleic acid molecule encoding the amino acid sequence of positions 21-234 or positions 1-234 in SEQ ID NO: 4; or
[0069] A nucleic acid molecule comprising the nucleotide sequence of positions 58-1875 or positions 1-1875 in SEQ ID NO: 1 and optionally a stop codon, and a nucleic acid molecule comprising the nucleotide sequence of positions 61-702 or positions 1-702 in SEQ ID NO: 3 and optionally a stop codon; or
[0070] A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 and a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 3.
[0071] In some other embodiments, when the fusion protein is Cetuximab-TNFα, correspondingly, said nucleic acid construct or combination of nucleic acid constructs may comprise or consist of the following nucleic acid molecules:
[0072] A nucleic acid molecule encoding the amino acid sequence of positions 17-621 or positions 1-621 in SEQ ID NO: 20, and a nucleic acid molecule encoding the amino acid sequence of positions 17-230 or positions 1-230 in SEQ ID NO: 22; or
[0073] A nucleic acid molecule comprising the nucleotide sequence of positions 49-1863 or positions 1-1863 in SEQ ID NO: 19 and optionally a stop codon, and a nucleic acid molecule comprising the nucleotide sequence of positions 49-690 or positions 1-690 in SEQ ID NO: 21 and optionally a stop codon; or
[0074] A nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 19 and a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 21.
[0075] As said above, a nucleic acid construct may comprise or consist of a vector. Accordingly, the present invention also provides a vector comprising an encoding nucleic acid molecule according to the present invention. In some embodiments, the vector may be an expression vector, which provides expression of the fusion protein in a host cell. In the context of present application, the term “vector” includes, for example, expression vectors, cloning vectors, plasmids, cosmids and viral vectors. Representative examples include, but are not limited to, vectors providing expression in eukaryotic cells (e.g., CHO cells and COS cells), vectors providing expression in Saccharomyces cerevisiae or Pichia pastoris cells, vectors providing expression in insect (e.g., silkworm) cells, and prokaryotic expression vector. In the context of the present invention, any vectors known in the art, such as those commercially available ones, can be used. For example, a nucleotide sequence encoding the novel fusion protein of the invention can be cloned into a vector as operably conjugate to a regulatory sequence for expression to thereby form an expression vector for the fusion protein.
[0076] Also provided herein is a host cell which is transformed to comprise the nucleic acid construct or combination of nucleic acid constructs as described above. In the context of the present application, the term “host cell” includes prokaryotic cells and eukaryotic cells. In some embodiments, the host cell may be selected from the group consisting of bacteria, fungi, yeasts, plant cells and animal cells. Examples of commonly used prokaryotic host cells include, for example, Escherichia coli and Bacillus subtilis. Commonly used eukaryotic host cells include yeast cells, insect cells and mammalian cells. In some embodiments, the host cell is a mammalian cell, such as a CHO cell, for example, CHO DG44 and CHO-K. In the context of the present application, the term “transform” refers to introducing a nucleic acid of interest or a vector comprising same into a host cell by any of the methods of transformation known in the art, whereby the obtained host cell is not a natural product. Methods of transformation may be selected according to types of the given host, and generally include, for example, electrotransformation, transfection using calcium chloride, DEAE-dextran or other reagents, particle bombardment, lipofection and infection (see Sambrook et al. Molecular Cloning Laboratory Guide, 2nd edition, 1989). A preferred method may be electrotransformation.
[0077] The present invention also provides a method for producing an antibody-TNFα fusion protein, wherein the method may include: culturing a transformed host cell as described above under a condition suitable for expression of the fusion protein to express the fusion protein; and isolating the fusion protein. The method may further include a step of subjecting culture of the host cell to a stress to obtain a strain having a high expression of the fusion protein, such as culturing under the stress of methotrexate, preferably an increasing gradient of methotrexate stress. The method may further include one or more steps of separating and/or purifying the fusion protein. Determination on parameters of culturing, such as medium, temperature and time, is within the normal skills of a person of skills in the art. Expression of the fusion protein can be detected using any of the conventional means of detection, such as SDS-PAGE and Western blotting. For purifying the fusion protein, any of the conventional separation and purification techniques can be used, including centrifugation, precipitation, filtration and chromatography. Specifically, useful chromatography techniques include, for example, affinity chromatography, gel filtration, ion exchange, hydrophobic chromatography and reverse chromatography. Specific examples include Protein-A affinity chromatography (e.g., POROS MabCapture A, Life Tech) and anion exchange chromatography (e.g., Q-600C, TOSOH).
[0078] Composition
[0079] A composition according to the present invention may comprise an effective amount of an antibody-TNFα fusion protein, a nucleic acid construct, a combination of nucleic acid constructs and/or a host cells according to the present invention. The composition may be a pharmaceutical composition, which may comprise a pharmaceutically acceptable carrier such as a solvent, excipient and adjuvant. In the context of the present application, the term “an effective amount” or “an effective dosage” refers to an amount providing to a human or animal subject a function or activity as intended and being tolerable to the human or animal subject. As used herein, the term “pharmaceutically acceptable” means that when a molecular or a composition is administered to an animal or human subject in an appropriate manner or route, it does not produce an undesired effect (such as toxicity, irritation and allergy), as demonstrated by a reasonable benefit/risk ratio. The term “a pharmaceutically acceptable carrier” is supposed to be compatible with the fusion protein of the invention, which can normally be blended with the protein without causing significant loss in efficacy of the pharmaceutical composition. Specific examples of substances that can be used as a pharmaceutically acceptable carrier or ingredient can be found in, for example, Remington: The Science and Practice of Pharmacy (2005), the 21st Century Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.
[0080] The pharmaceutical compositions according to the present invention may be formulated into a dosage form as appropriate for a specific dosage or manner of administration.
[0081] A nucleic acid molecule may be delivered to a subject in the form of a naked nucleic acid molecule, a co-delivery preparation or a recombinant vector (e.g., a plasmids or a viral vector) comprising and/or expressing the nucleic acid molecule. Suitable delivery reagents for administration include the lipophilic reagents in the Minis Transit TKO, lipofectins, lipofectamines, cellfectins, cationic polymeric reagents (e.g., polylysin), or liposomes, as well as any other means known to a person of skills in the art.
[0082] In some embodiments, the composition of the invention may further comprise one or more additional active agents for treating or preventing tumors. In some embodiments, the additional active agent for treating or preventing tumors are selected from chemotherapeutic agents and radiotherapeutic agents, such as alkylating agents, antimetabolites, antitumor antibiotics, botanical anticancer medicines, hormones and immunotherapeutics. Examples include mitosis inhibitors, transcription inhibitors and protein synthesis inhibitors, and more specifically, vinblastine, vincristine, vindesine, vinorelbine, colchicamine, colchicine, colchicamide, podophyllotoxin, etoposide, teniposide, paclitaxel or docetaxel, camptothecin, homoharringtonine, procarbazine, asparaginase, cisplatin, carboplatin, mitoxantrone, tamoxifen, cyclophosphamide, chlorambucil hydrochloride, lomustine, semustine, cetepa, busulfan, methamphetamine, chlorambucil, fluorouracil, Tegafur, efluidin, carmofur, mercaptopurine, methotrexate, cytarabine, cyclocytidine, mercaptoguanine, hexamethylmelamine, hydroxyurea, mitomycin, doxorubicin (adriamycin), epirubicin, bleomycin, pelomycin, atorvastatin, gleevec, gemcitabine, actinomycin D, cycloheximide, topotecan and/or leuprolide; especially actinomycin D, cycloheximide, doxorubicin; and analogs, derivatives, prodrugs or metabolites thereof. The present invention found that such a composition provides an effect of synergism in inducing apoptosis in tumor cells, inhibiting growth of tumor and tumor treatment. This is unexpected.
[0083] In the combination, the antibody-TNFα fusion protein may, for example, be present at an amount of at least 10.sup.−3 ng/ml, at least 10.sup.−2 ng/ml, at least 10.sup.−1 ng/ml, at least 1 ng/ml, at least 10 ng/ml, at least 10.sup.2 ng/ml, at least 10.sup.3 ng/ml, at least 10.sup.4 ng/ml, at least 10.sup.5 ng/ml, at least 20 ng/ml, at least 40 μg/ml, or a range formed from any two of the above as the lower and upper limits, such as 20 ng/ml to 40 μg/ml, less than 30 ng/ml, less than 20 ng/ml; or an amount of at least 10.sup.−5 nM, at least 10.sup.−4 nM, at least 10.sup.−3 nM, at least 10.sup.−2 nM, at least 10.sup.−1 nM, at least 1 nM, at least 10 nM, at least 10.sup.2 nM, or a range formed from any two of the above as the lower and upper limits.
[0084] In the combination, the content of the one or more additional agents for treating or preventing tumors can be determined according to the relevant guidelines of medication in clinic and other conventional dosages. Or, the content may be, for example, at least 1 nM, at least 10 nM, at least 15 nM, at least 20 nM, at least 100 nM, at least 1 μM, at least 2 μM, at least 3 μM, at least 4 μM, at least 5 μM, at least 10 μM, at least 20 μM, at least 30 μM, at least 40 μM, at least 50 μM, at least 100 μM, or a range formed from any two of the above as the lower and upper limits; or at least 1 μg/ml, at least 2 μg/ml, at least 3 μg/ml, at least 4 μg/ml, at least 5 μg/ml, at least 6 μg/ml, at least 7 μg/ml, at least 8 μg/ml, at least 9 μg/ml, at least 10 μg/ml, or a range formed from any two of the above as the lower and upper limits.
[0085] In context of the composition, the fusion protein and the additional active agent may be present at a ratio equivalent to the ratio between any one of the amounts specified for the fusion proteins and any one of the amounts specified for the active agent as in the above. For example, the ratio may be equivalent to: 1-100 ng/ml to 1 nM-100 μM; such as 1-100 ng/ml to 1-20 nM, 1-100 ng/ml to 15 nM, 1 ng/ml to 1-20 nM, 100 ng/ml to 1-20 nM, 1 ng/ml to 15 nM, 100 ng/ml to 15 nM; such as 1-100 ng/ml to 1-100 μM, 1-100 ng/ml to 3-50 μM, 1-100 ng/ml to 4-40 μM, 1-100 ng/ml to 10-30 μM, 1 ng/ml to 1-100 μM or 3-50 μM or 4-40 μM or 10-30 μM, 100 ng/ml to 1-100 μM or 3-50 μM or 4-40 μM or 10-30 μM, such as 1 ng/ml to 33 μM, 100 ng/ml to 33 μM, 1 ng/ml to 11 μM, 100 ng/ml to 11 μM, 1 ng/ml to 3.7 μM, 100 ng/ml to 3.7 μM; or such as 10.sup.−3-10.sup.5 ng/ml to 1-10 μg/ml, 10.sup.−3-10.sup.5 ng/ml to 5 μg/ml, 10.sup.−3-10.sup.2 ng/ml to 5 μg/ml, 10.sup.−2-10.sup.2 ng/ml to 5 μg/ml. The phrase “equivalent to” here means that the ratio is the same, while the specific amounts of the two agents are not limited to the values as listed.
[0086] Notably, the term “composition” means that the ingredients act in cooperation, and it does not necessarily mean that the ingredients are always in the same mixture or system. For example, ingredients of a composition may be physically or spatially separated from one another, may be administered to a subject at the same time point after being mixed or not mixed, or sequentially administered to a subject at different time points, as long as they act in cooperation at the site of target. For example, according to the present application, embodiments of the composition include a kit, which may comprise one or more ingredients of the composition of the invention in one or more containers.
[0087] Applications
[0088] The antibody-TNFα fusion protein, the nucleic acid construct(s), the vector, the host cell and the composition according to the present invention can be used to induce apoptosis in tumor cells, to inhibit growth of tumor, to prevent and/or treat tumors, and can also be used for manufacturing a product, such as a medicament, a pharmaceutical formulation, a kit, etc., for these purposes.
[0089] In the context of the present application, examples of tumors include those expressing tumor-specific antigens or have high expression of tumor-associated antigens, including but not limited to tumors that are responsive to TNFα treatment. Examples of tumor-specific antigens and tumor-associated antigens include but are not limited to: ErbB family members, such as EGFR, Her-2, Her-3 and Her-4. Specifically, examples of tumors include, but are not limited to: gastric cancer, breast cancer, ovarian cancer, melanoma, bowel cancer such as colon and rectal cancer, liver cancer, pancreatic cancer, kidney cancer, lung cancer, head and neck cancer, cervical cancer, skin cancer, esophageal cancer, bone cancer and leukemia.
[0090] The antibody-TNFα fusion protein, the nucleic acid construct(s), the vector, the host cell and the composition according to the present invention can also be used in combination with an additional active agent or therapy for treating or preventing tumors. The additional active agent for treating or preventing tumors is as described in the section of “composition” above. The additional therapy may be, for example, surgery, radiotherapy and chemotherapy.
[0091] As for dosage of the fusion protein of the invention, a physician knows to determine a dosage appropriate for a patient depending on known factors including, for example, species, race, age, bodyweight, disease status and administration manner. For example, the dosage may be at least 0.1 mg/kg bodyweight, at least 0.3 mg/kg bodyweight, at least 1 mg/kg bodyweight, or at least 3 mg/kg bodyweight. The fusion protein or the composition of the present invention can be administered in various ways as appropriate, including oral administration and parenteral administration, such as injection and infusion.
[0092] As can be understood by a person of skills in the art, the features and approaches in the present disclosure can be recombined without departing from the spirit and scope of the present invention. More aspects of the invention would become obvious to a person of skills in the art from the present disclosure.
EXAMPLES
[0093] The present invention will be further illustrated with reference to the following specific examples. It will be appreciated by a person of skills in the art that various modifications and change can be made without departing from the spirit and scope of the invention.
[0094] In the following examples, unless otherwise specified, methods or processes can be conducted as conventional practiced in the art, for example, as described in “Molecular Cloning: A laboratory Manual” (3rd edition, 1989, Cold Spring Harbor Laboratory Press, New York), or by following manufacturer's instruction. DNA sequencing can be conducted using known methods or purchased from a service provider.
Example 1: Construction of Expression Plasmid for Antibody-TNFα Fusion Protein
[0095] 1. Construction of Expression Plasmid for her-2 Antibody-TNFα Fusion Protein
[0096] Trastuzumab was used as an example of Her-2 antibodies. The complete cDNAs encoding the heavy chain and the light chain of Trastuzumab were synthesized by GenScrip (USA), and were cloned into vector pUC57. The cDNA of human TNFα was obtained from OpenBiosystems (USA).
[0097] It has been shown in quite some reports that in expression and preparation of monoclonal antibodies, deletion of lysine residue(s) at C-terminal of heavy chains is common. Therefore, we removed this lysine residue to ensure integrity of the antibody fusion protein.
[0098] The gene encoding the heavy chain of Trastuzumab and the gene encoding TNFα was connected by a two-step polymerase chain reaction (PCR) process. In the first step, the gene of the heavy chain was amplified using synthetic DNA primers by PCR (High-Fi Polymerase Pfx, Invitrogen):
[0099] 5′-primer M13-F (SEQ ID NO: 5): 5′-TGTAAAACGACGGCCAGT-3′, on vector;
[0100] 3′-primer KDP004 (SEQ ID NO: 6): 5′-TCCTGGGGACAGTGACAGTG-3′, specific for the antibody heavy chain gene.
[0101] The gene encoding the TNFα moiety was amplified by PCR in the same way:
TABLE-US-00001 5′-primer KDP045 (SEQ ID NO: 7): 5′-CACTGTCACTGTCCCCAGGAGTCAGATCATCTTCTCGAACC-3′; 3′-primer BGH-R (SEQ ID NO: 8): 5′-AACTAGAAGGCACAGTCGAGGC-3′, on vector.
[0102] Therein, sequence of the first 20 nucleotides in the TNF-F primer was complementary to the nucleotide sequence in the Her2-R primer, so that the two fragments from PCR could be connected in the second step of overlap extension PCR.
[0103] The two resultant PCR fragments were purified using DNA Gel Purification (Tiangen Biotech (Beijing) Co., Ltd.) before the second step of overlap PCR. The purified DNA fragments were 3-times diluted with eluent to facilitate ligation via overlap PCR:
TABLE-US-00002 5′-primer: still M13-F (SEQ ID NO: 5); 3′-primer BGH-R (SEQ ID NO: 8): 5′-TGGTGGTGTCTAGAGACTCACAGGGCAATGATCCC-3′,
containing an XbaI restriction site for cloning.
[0104] A NotI restriction site was placed upstream to the transcription initiation site of the gene of Trastuzumab heavy chain. The fragment obtained from overlap PCR was gel purified and subjected to NotI/XbaI double digestion (Takara). The digested PCR fragment was cloned into the correspondingly digested expression vector for mammalian cells. The expression vector for mammalian cells was a modified pcDNA3.1 plasmid (Invitrogen), wherein the anti-neomycin gene was replaced by DHFR (dihydrofolate reductase) gene. The modified vector is useful for screening for mammalian cells having high expression of stably exogenous proteins. The obtained recombinant plasmid was transfected into competent bacterial strain DH5a. Positive colonies containing the correct recombinant plasmid were identified by colony PCR. The recombinant plasmid was purified, and correct sequence of the recombinant gene of Trastuzumab heavy chain-TNFα was confirmed by restriction digestion and sequencing.
[0105] The cDNA of Trastuzumab's light chain was sub-cloned into another pcDNA3.1 plasmid using enzymes NotI and XbaI.
[0106] 2. Construction of Expression Plasmid for EGFR Antibody-TNFα Fusion Protein
[0107] Cetuximab was used as an example of EGFR antibodies. The gene encoding the heavy chain variable region of Cetuximab (VH, with signal peptide) and the gene encoding the light chain variable region of Cetuximab (VL, with signal peptide) were synthesized by Genewiz (Beijing) Co. Ltd., and were cloned into vector pUC57.
[0108] To construct the gene of Cetuximab heavy chain-TNFα, first, the Cetuximab VH gene and IgG1 Fc gene were connected by PCR to obtain gene of a complete heavy chain of Cetuximab; and then, the obtained gene of Cetuximab heavy chain was connected with the TNFα gene by PCR to obtain the gene of Cetuximab heavy chain-TNFα. The obtained PCR fragment was digested with NotI and XbaI, and cloned into correspondingly digested expression vector for mammalian cells. In this example, the expression vector for mammalian cells was a modified pcDNA3.1 plasmid (Invitrogen) wherein the anti-neomycin gene was replaced by the gene of rat glutamine synthetase (GS).
[0109] The primers for PCR amplification of Cetuximab VH gene were M13-R (Forward, 5′-CAGGAAACAGCTATGACC, SEQ ID NO: 9) and KDP077 (Reverse, 5′-GCTAGGCCCCTTTGTTGATGCGGCGGACACGGTCACGAGGG, SEQ ID NO: 10). The primers for amplifying IgG1 Fc fragment were KDP020 (5′-GCATCAACAAAGGGGCCTAGC, SEQ ID NO: 11) and BGH-R (5′-AACTAGAAGGCACAGTCGAGGC, SEQ ID NO: 12).
[0110] The PCR fragment of Cetuximab VH gene and the fragment of IgG1 Fc were connected by overlap extension PCR using primer KDP092 (Forward, 5′-ATGGAACGCGGCCGCCACC, SEQ ID NO: 13) and primer KDP093 (Reverse, 5′-TCTAGCATTTAGGTGACAC, SEQ ID NO: 14). The obtained fragment of full-length heavy chain was cloned into said expression vector using restriction enzymes NotI/XbaI.
[0111] Then gene encoding Cetuximab heavy chain-TNFα was cloned according to the procedure as described for the gene encoding Trastuzumab heavy chain-TNFα. The PCR primers for amplifying the gene of Cetuximab heavy chain were primer CMV-P (Forward, 5′-CGCAAATGGGCGGTAGGCGTG, SEQ ID NO: 15) and primer KDP004 (SEQ ID NO: 6). The primers for amplifying the TNFα gene were KDP045 (SEQ ID NO: 7) and BHG-R (SEQ ID NO: 12). The two genes were connected using PCR primers KDP033 (5′-AGCTTGGTACCCTCGAGG, SEQ ID NO: 16) and KDP093 (SEQ ID NO: 14). The obtained gene of Cetuximab heavy chain-TNFα was cloned into said expression vector using restriction enzymes NotI/XbaI.
[0112] To construct gene of the Cetuximab light chain, the gene of light chain variable region (VL) and the gene of constant region (kappa) were amplified by PCR. The PCR primers for the VL gene were M13-R (SEQ ID NO: 9) and KDP094 (GACAGATGGAGCGGCCACAGTTCGCTTCAGCTCCAGCTTTGTTCC, SEQ ID NO: 17). The PCR primers for the kappa gene were KDP010 (ACTGTGGCCGCTCCATCTGTC, SEQ ID NO: 18 and BGH-R (SEQ ID NO: 12). The PCR fragment of Cetuximab VL gene and the PCR fragment of the kappa gene were connected by overlap extension PCR using primer M13-R (SEQ ID NO: 9) and primer KDP093 (SEQ ID NO: 14). The obtained fragment of the full-length light chain gene was cloned into said expression vector using NotI/XbaI restriction enzymes.
Example 2: Establishment of Cell Line Stably Expressing Antibody-TNFα Fusion Protein
[0113] The host cell CHO DG44 was obtained from Invitrogen. The cells were cultured and subcultured according to the CHO DG44 manual from Invitrogen. Non-transfected cells were suspension cultured in CD DG44 medium (Invitrogen) supplemented with 8 mM L-glutamine (Sigma) and 5 μg/ml recombinant human insulin. CHO-K cell was obtained from the Cell Bank of the Type Culture Collection Committee of Chinese Academy of Sciences (Shanghai). The primary cells exhibited adherent growth in a serum-supplemented medium, and were habituated to be capable of suspension growth in a serum-free medium (chemically defined medium, abbr. “CD”). In this example, the CHO DG44 cells were used to express the Trastuzumab-TNFα fusion protein, and the CHO-K cells were used to express the Cetuximab-TNFα fusion protein.
[0114] Briefly, the strain stably expressing the Trastuzumab-TNFα fusion protein was constructed as follows. The expression plasmid of the fusion protein's heavy chain and the expression plasmid of the fusion protein's light chain were prepared using the Plasmid Maxi Preparation Kit (TIANGEN, China). 100 μg pcDNA3.1 comprising the antibody heavy chain-TNFα and 100 μg pcDNA3.1 comprising the antibody light chain prepared according to Example 1 were linearized by digestion using restriction endonuclease EcoRI. The DG44 cells were subcultured for at least three passages before transfection. 1×10.sup.7 DG44 cells were mixed with the digested plasmids in 0.8 ml CD DG44 growth medium and then transferred in to a 0.4-cm pulser (Bio-Rad), wherein the cell/plasmid mixture was pulsed on Gene Pulser Xcell (Bio-Rad). The transfected cells were then cultured in a T-75 square flask containing 20 ml growth medium for 24 hours at 37° C. in 8% CO.sub.2 in incubator.
[0115] After the 24-hour incubation, the transfected cells were plated on a 96-well plate by limiting dilution, using the OptiCHO medium as the medium for screen, wherein the medium was supplemented with 8 mM glutamine, 5 μg/ml recombinant human insulin and 100 nM methotrexate (MTX, Sigma). The cells were incubated at 37° C. in 8% CO.sub.2 in incubator. 3 weeks later, culture liquid from the wells observed with colony formation was examined by ELISA using alkaline phosphatase-conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch Lab), whereby clones with the highest expression were picked and amplified, then assayed via ELISA and amplified again, which finally ended up with 12 cell lines with highest stable expression.
[0116] The 12 clones were cultured under stress using an increasing gradient of MTX concentrations for enhanced expression. Totally three rounds of incubation under stress (500 nM, 2 μM and 10 μM of MTX) were conducted, each for about 3 weeks. After the 3-rounds stress incubation, the 12 clones were tested for antibody expression, whereby 4-5 cell lines with increased antibody expression were obtained.
[0117] Without experiencing the MTX stress, the cell lines with stable antibody expression were low in expression level. After 3 rounds of stress treatment, with the MTX magnitude increasing from 0.1 μM to 10 μM in the culture medium, the antibody expression was significantly increased in most of the cell lines exposed to the stress. From these cell lines, one strain (#1G5) was pick and plated on a 96-well plate and cultured by limiting dilution to obtain a monoclonal strain with high expression of the antibody fusion protein. Cell strains stably expressing Cetuximab-TNFα fusion protein was constructed by the method and steps outlined as follows. CHO-K cells were co-transfected with plasmid encoding the chain of Cetuximab heavy chain-TNFα and plasmid encoding Cetuximab light chain via pulse-transfection. The transfected cells were plated on a 96-well plate and cultured by limiting dilution in medium containing L-aminosulfoxide methionine (MSX) to screen for strains stably transfected with the plasmids. Cell lines with positive antibody expression were picked by ELISA. Specifically, supernatant comprising said cells were added onto a ELISA plate coated with recombinant human EGFR protein; antibody was detected using alkaline phosphatase (AP) conjugated goat anti-human IgG1 Fc antibody; cell lines having high expression were picked and amplified, then subjected to the next round of screening; and thereby, CHO-K cell lines with stable and high expression of Cetuximab-TNFα fusion protein were obtained.
Example 3: Preparation, Purification and Identification of Antibody-TNFα Fusion Protein
[0118] The cell line with high expression of antibody-TNFα obtained according to Example 2 was cultured and amplified to 2 liters. Supernatant of the culture was harvested for purification and preparation of the antibody. The purification was Protein-A affinity chromatography (POROS MabCapture A, Life Tech) followed by anion exchange chromatography (flow through).
[0119] The purified antibody was analyzed by reducing and non-reducing SDS-PAGEs and HPLC-SEC (High Pressure Liquid—Molecular Sieve, TSKgel G3000SWXL, TOSOH Bioscience).
[0120] Results and Analysis
[0121] As shown by the gel of non-reducing SDS-PAGE in
[0122] The HPLC-SEC result of the Trastuzumab-TNFα fusion protein was shown in
[0123] Non-reducing SDS-PAGE, reducing SDS-PAGE and HPLC-SEC results of the Cetuximab-TNFα fusion protein were the same, as shown in
Example 4: Tumor Antigen Binding of Antibody-TNFα Fusion Protein
[0124] Binding of the prepared Trastuzumab-TNFα fusion protein to extracellular domain (ECD) of the membrane protein Her-2 was studied via ELISA, in comparison with the antibody Trastuzumab (CTTQ Pharmaceutical Group Co., Ltd).
[0125] 25 nM recombinant human Her-2 ECD (donated by ANKEBIO (Group) Co., Ltd.) was dissolved in 50 mM NaHCO.sub.3 (pH 9.6). The recombinant protein (50 μL) was added into wells of a 96-well ELISA plate and was stored at 4° C. overnight in refrigerator. The next day, the ELISA plate was washed 3 times with PBST (PBS with 0.05% Tween-20), and then blocked with 100 μl/well blocking buffer being PBST comprising 3% BSA. The ELISA plate was incubated at 37° C. for 1 hour in incubator. The Trastuzumab-TNFα fusion protein and the Trastuzumab antibody were each prepared into 3-fold serial dilutions in binding buffer being PBST with 1% BSA. The blocking buffer was drained, and the fusion protein and Trastuzumab in the 3-fold serial dilutions were added (50 μL/well) to react at 37° C. for 1 hour in incubator. The dilutions were removed, the ELISA plates were washed 3 times with PBST, and the secondary antibody (alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab) was added (50 μL/well) to react at 37° C. for 1 hour in incubator. The staining antibody was removed, PBST wash buffer was added (200 μL/well) and the ELISA plates were placed on an orbital shaker at 100 rpm for 5 minutes; then, the wash buffer was removed and the washing steps were repeated 4 times. The developer solution (PNPP) was added (50 μl/well) and the ELISA plates were incubated at 37° C. in incubator for development. The plates were read on a microplate reader at 405 nm/655 nm.
[0126] Binding of the prepared Trastuzumab-TNFα fusion protein to the Her-2 surface protein was also assayed via flow cytometry. Three Her-2 positive human tumor cell lines, i.e., human pancreatic cancer cell line PANC-1, human ovarian cancer cell line SKOV-3 and human breast cancer cell line SKBR-3 (all from Cell Bank of the Chinese Academy of Sciences) were used. The cells were prepared in pre-cooled FACS working buffer (PBS with 0.1% FBS) at 3×10.sup.6 cells/ml and divided into aliquots of 100 μl/tube, which were blocked on ice for 1 hour. The Trastuzumab-TNFα fusion protein and Trastuzumab were each diluted to 10 μg/ml in the FACS working buffer. The dilutions (10 μL) were added into cell suspension (100 μL) and incubated on ice for 30 minutes. At the end of incubation, to each tube, FACS working buffer (1 ml) was added and mixed with the cell suspension by vortex; the cell suspension was then centrifuged at 1200 rpm for 5 minutes; the supernatant was discarded and the washing steps were repeated once. FITC-labeled goat anti-human IgG Fc antibody (Jackson ImmunoResearch Lab) was diluted in the FACS working buffer, and then added into the cell suspension (10 μL per tube) at a final concentration of 1 μg/ml. The tubes were incubated on ice for 30 minutes in dark. At the end of incubation, to each tube, FACS working buffer (1 ml) was added and mixed with the cell suspension by vortex; the cell suspension was then centrifuged at 1200 rpm for 5 minutes; the supernatant was discarded and the washing steps were repeated once. The cells were assayed on flow cytometer C6 (BD Biosciences).
[0127] Results and Analysis
[0128] As shown by the ELISA result in
[0129] As shown by the flow cytometry results in
Example 5: Binding of Antibody-TNFα Fusion Protein to TNFR In Vitro
[0130] Binding of the Trastuzumab-TNFα fusion protein to the extracellular domain of human TNFR2 (sTNFR) was studied via ELISA. The fusion protein of sTNFR2 and IgG Fc (Celgen Biopharmaceutical Co., Ltd.) was diluted to 1 nM in 50 mM NaHCO.sub.3 (pH 9.6). The solution (50 μL) was added into the wells of a 96-well ELISA plate and was stored at 4° C. overnight in refrigerator. The next day, the ELISA plate was washed 3 times with PBST (PBS with 0.05% Tween-20), and then blocked with 100 μl/well blocking buffer being PBST comprising 3% BSA. The ELISA plate was incubated at 37° C. for 1 hour in incubator. The Trastuzumab-TNFα fusion protein obtained according to Example 3 was prepared into 4-fold serial dilutions in binding buffer being PBST+1% BSA. The blocking buffer was drained, and the fusion protein in the 4-fold serial dilutions was added (50 μL/well) to react at 37° C. for 1 hour in incubator. The dilutions of fusion protein were removed, the ELISA plates were washed 3 times with PBST, and the secondary antibody (alkaline phosphatase-conjugated goat anti-human IgG Fab antibody, Jackson ImmunoResearch Lab) was added (50 μL/well) to react at 37° C. for 1 hour in incubator. The staining antibody was removed, PBST wash buffer was added (200 μL/well) and the ELISA plates were placed on an orbital shaker at 100 rpm for 5 minutes; then the wash buffer was removed and the washing steps were repeated 4 times. The developer solution (PNPP) was added (50 μl/well) and the ELISA plates were incubated at 37° C. in incubator for development. The plates were read on a microplate reader at 405 nm.
[0131] Results and Analysis
[0132] As shown in
Example 6: Apoptosis in L929 Cells Induced by Antibody-TNFα Fusion Protein
[0133] The Trastuzumab-TNFα fusion protein and the Cetuximab-TNFα fusion protein were tested on cytotoxicity in mouse fibroblasts L929, in comparison with recombinant human TNFα protein.
[0134] L929 cells (Cell Bank of the Chinese Academy of Sciences (Shanghai)) were cultured in RPMI1640 medium with 10% FBS (Invitrogen). The day before experiment, the L929 cells (10000 cells/well) were added onto a 96-well plate and incubated at 37° C. in a 5% CO.sub.2 in incubator. The next day, the antibody-TNFα fusion protein and the recombinant human TNFα in 3-fold serial dilutions in the cell growth medium were added into the cells to final concentrations from 0 to 1.18 nM (totally ten concentrations). After incubation for another 20 hours, the cells were stained with crystal violet, and the plates were read on a microplate reader at 570 nm.
[0135] Results and Analysis
[0136] As shown in
Example 7: Cytotoxicity of her-2 Antibody-TNFα Fusion Protein on her-2 Positive Human Gastric Cancer Cells
[0137] Human gastric cancer cell NCI-N87 with high expression of Her-2 protein is less sensitive to Trastuzumab. Trastuzumab induces moderate apoptosis in NCI-N87 cells only at fairly high concentrations. To study activity of the Her-2 antibody-TNFα fusion protein of the invention in inducing apoptosis in NCI-N87 cells, (1) the Trastuzumab-TNFα fusion protein, (2) a (Trastuzumab+TNFα) mixture and (3) TNFα were compared for their impacts on viability in NCI-N87 cells.
[0138] The Her-2 positive human gastric cancer cells NCI-N87 (Cell Bank of the Chinese Academy of Sciences (Shanghai)) were cultured and subcultured in RPMI medium 1640 supplemented with 10% FBS (Gibco). The day before experiment, the cells (lx 10.sup.6 cells/well) in growth medium (150 μl) were added onto a 96-well plate. The next day, the Trastuzumab-TNFα fusion protein, TNFα and the Trastuzumab+TNFα mixture (PeproTech, USA) (a mixture with the total amount of proteins substantially equivalent to that of the antibody-TNFα fusion protein by weight, with the antibody to TNFα (dimeric) ratio by weight being 5:1, approximately 2 molecules of monomeric TNFα per antibody molecule) prepared in serial dilutions in the growth medium were added (50 μl/well) into the cells on the 96-well plate, mixed and cultured for another 60 hours in incubator. Cell viability was detected using MTT assay and cell viability using CCK-8.
[0139] Results and Analysis
[0140] First, we examined and compared the effects in killing NCI-N87 between the Trastuzumab-TNFα fusion protein and the Trastuzumab antibody. Three stages of effects were observed with the Trastuzumab-TNFα fusion protein in NCI-N87 cells: at concentrations below 30 ng/ml, the fusion protein effectively induced apoptosis in cells; at concentrations between 20 ng/ml and 40 μg/ml, the killing decreased with increase in concentration; and at concentrations above 40 μg/ml, the killing increased with increase in concentration (data not shown). Trastuzumab did not substantially impact survival of NCI-N87 cells at all concentrations in test (data not shown).
[0141] Next, we studied killing in NCI-N87 cells by the fusion protein at low concentrations, in comparison with TNFα. As shown in
[0142] Then, we studied the effect of the TNFα+Trastuzumab mixture on survival in NCI-N87 cells. NCI-N87 cells were treated with a mixture prepared by mixing 2 ng/ml TNFα and 10 ng/ml Trastuzumab, which corresponded to 10 ng/ml of Trastuzumab-TNFα fusion protein. Apoptosis was detected, in comparison with 10 ng/ml of the Her-2 antibody-TNFα fusion protein and with 2 ng/ml of TNFα. As shown in
[0143] There results suggest that the Her-2 antibody-TNFα fusion protein of the invention has a unique profile of effects on cell survival, and exhibits an effect of synergism as compared with TNFα alone, and a simple blend of the Her-2 antibody and TNFα.
Example 8: Cytotoxicity of EGFR Antibody-TNFα Fusion Protein on EGFR Positive Human Gastric Cancer Cells
[0144] Human gastric cancer cell NCI-N87 expresses EGFR protein. To study activity of the EGFR antibody-TNFα fusion protein of the invention in inducing apoptosis in NCI-N87 cells, (1) the Cetuximab-TNFα fusion protein and (2) a mixture of Cetuximab mAb+TNFα were compared for impacts on viability in NCI-N87 cells.
[0145] EGFR-positive human gastric cancer cells NCI-N87 (Cell Bank of the Chinese Academy of Sciences (Shanghai)) were cultured and subcultured in RPMI medium 1640 supplemented with 10% FBS (Gibco). The day before experiment, the cells (1×10.sup.6 cells/well) in growth medium (150 μl) were added onto a 96-well plate. The next day, the Cetuximab-TNFα fusion protein and the Cetuximab+TNFα mixture (PeproTech, USA) prepared in serial dilutions in the growth medium were added (50 μl/well) into the cells on the 96-well plate, mixed and cultured for another 48 hours in incubator. Cell viability was assayed using CCK-8.
[0146] Results and Analysis
[0147] As shown in
Example 9: Synergistic Induction of Apoptosis by her-2 Antibody-TNFα Fusion Protein Combined with Anti-Tumor Chemotherapeutic in her-2 Positive Human Tumor Cells
[0148] In the following, the Her-2 positive tumor cells were all obtained from the Cell Bank of the Chinese Academy of Sciences. BT474 cell and NCI-N87 cells were cultured in RPMI 1640 medium, SKBR-3 in DMEM (Invitrogen) and SKOV-3 in McCOY's 5A medium (Invitrogen), all these media contained 10% FBS. The cells were incubated in the media added with different chemotherapeutics at different concentrations for 2 days: actinomycin D (MedChem Express) and doxorubicin (MedChem Express); then, the Trastuzumab-TNFα fusion protein was added at different concentrations and the cells were incubated for another 20 hours. Cell viability was detected by MTT assay.
[0149] Results and Analysis
[0150] As shown, an effect of synergism was observed with both the combination of Trastuzumab-TNFα fusion plus actinomycin D (
[0151] As shown in
[0152] The Trastuzumab-TNFα fusion protein in combination with doxorubicin exhibited a fairly strong effect of synergism in inducing apoptosis in NCI-N87 and BT-474 cells. 1 ng/ml of Trastuzumab-TNFα fusion protein in combination with 33 μM or 11 μM doxorubicin induced apoptosis in 80% NCI-N87 and BT-474 cells, while they had little effect on viability in these cells at said concentrations when being used alone (
Example 10: Construction of Mouse Tumor Cells Stably Expressing Human EGFR or Human Her-2
[0153] Mouse melanoma cells B16, mouse colon cancer cells CT26 and mouse forestomach carcinoma (MFC) cells were all obtained from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences. The cells were cultured in RPMI1640/10% FBS medium (Gibco).
[0154] The gene for human Her-2 expression was cloned in the expression vector pcDNA3.1 (Invitrogen); the recombinant plasmid was transfected into the three mouse tumor cells using Lipofectamine 3000 (Invitrogen); the transfected cells were cultured in RPMI/10% FBS medium supplemented with G418 (Sigma); and, stably transfected cells were obtained thereby. The stably transfected cells were sorted using a flow cytometer (Influx, BD Biosciences), whereby the monoclonal stable B16/Her-2, CT26/Her-2 and MFC/Her-2 cell lines with high Her-2 expression were picked.
[0155] The gene for human EGFR expression was cloned in the expression vector pCMV3 (Sino Biological Inc); the recombinant plasmid was transfected into the B16 mouse tumor cells and MFC mouse tumor cells using Lipofectamine 3000 (Invitrogen); the transfected cells were cultured in RPMI/10% FBS medium supplemented with Hygromycin B (Sigma); and, stably transfected cells were obtained thereby. The stably transfected cells were sorted using a flow cytometer (Influx, BD Biosciences), whereby the monoclonal stable B16/EGFR and MFC/EGFR cell lines with high EGFR expression were picked.
Example 11: Transcription Inhibitor and Protein Synthesis Inhibitor Enhancing Toxicity of her-2 Antibody-TNFα Fusion Protein in Mouse Forestomach Carcinoma Cells Expressing Human her-2 (MFC/her-2)
[0156] Toxicity of the Her-2 antibody-TNFα fusion protein on the MFC/Her-2 cells in the presence of actinomycin D (ActD, a transcription inhibitor) or cycloheximide (CHX, a protein synthesis inhibitor) was assayed, in comparison with the toxicity in absence of ActD and CHX. The MFC/Her-2 cells were plated on a 96-well plate and cultured for 1 day, then the medium was drained and to different wells were added different concentrations of the Trastuzumab-TNFα fusion protein in combination with ActD (5 μg/ml) or CHX (μg/ml) at identical concentrations. The cells were incubated for another 20 hours. The toxicity of the antibody-TNFα proteins on cells was detected by counting living cells using the Cell Counting Kit (CCK-8). The controls were the Trastuzumab+TNFα protein mixture, wherein the total amount of proteins was substantially equivalent to that in the Her-2 antibody-TNFα fusion protein, with the antibody to TNFα (dimeric) ratio by weight being 5:1, approximately 2 molecules of monomeric TNFα per antibody molecule as in the Her-2 antibody-TNFα fusion protein molecule.
[0157] The described experiment was repeated using wild-type (wt) MFC cells, and the result was compared with that of the MFC cells expressing human Her-2.
[0158] Results and Analysis
[0159] As shown in
[0160] As shown in
[0161] As shown in
Example 12: EGFR Antibody-TNFα Fusion Protein in Combination with Transcription Inhibitor Inducing Synergistic Effect of Apoptosis in Mouse Forestomach Carcinoma Cells Expressing Human EGFR (MFC/EGFR)
[0162] The EGFR antibody-TNFα fusion proteins and the monoclonal antibody+TNFα mixture were compared on cytotoxicity in the MFC/EGFR cells in the presence of transcription inhibitor ActD at 5 μg/ml. In the Cetuximab+TNFα mixture, the ratio between the two molecules was as specified in Example 7. At the same time, cytotoxicity of the Cetuximab-TNFα fusion protein in absence of ActD was examined. The cells were incubated with said antibodies for 20 hours, and then cell viability was detected using Cell Counting Kit CCK-8.
[0163] Results and Analysis
[0164] As shown in
Example 13: EGFR Antibody-TNFα Fusion Protein in Combination with Transcription Inhibitor Synergistically Inducing Apoptosis in Mouse Melanoma Cells Expressing Human EGFR
[0165] The EGFR antibody-TNFα fusion protein and the Cetuximab+TNFα mixture were compared on cytotoxicity in B16/EGFR cells in the presence of transcription inhibitor ActD at 5 μg/ml. The formulation of the Cetuximab+TNFα mixture was as specified in Example 7. Also examined is cytotoxicity of the EGFR antibody-TNFα fusion protein in absence of ActD. The cells were incubated with said antibodies for 20 hours, and then cell viability was detected using Cell Counting Kit CCK-8.
[0166] Results and Analysis
[0167] As shown in
Example 14: In Vivo Anti-Tumor Activity of her-2 Antibody-TNFα Fusion Protein on her-2-Positive Mouse Melanoma B16 in Mice
[0168] The C57BL/6 mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. The animals were maintained in SPF environment.
[0169] Twenty 6-7 weeks old male C57/B6 mice were divided into 4 groups of 5 animals/group. The animals were subcutaneously injected with B16/Her-2 cells (1×10.sup.6 cells/animal) at axilla. When the tumor reached 200-600 mm.sup.3, the mice were injected intravenously with the Trastuzumab-TNFα fusion protein or Trastuzumab at different dosages. There were two dosage groups for the Trastuzumab-TNFα fusion protein: 1 mg/kg and 3 mg/kg; one group for Trastuzumab: 3 mg/kg; and one group of control: injection of even volumes of PBS. The treatments were administrated twice a week for two weeks, i.e., totally of 4 dosings. Tumor volume was measured and animals weighed at each dosing. 4 days after the last dosing, the mice were sacrificed by cervical dislocation. Orbital vein blood was collected, tumor weight and spleen weight and size were recorded, and tumors were pictured via anatomy.
[0170] Results and Analysis
[0171] As shown in
[0172] Meanwhile, change in bodyweight was taken as a measurement of toxicity of the fusion protein in mouse. Weight of the animals dropped by about 10% three days after the first administration, then stayed and began to recover during the continuous administration. This suggests that the toxicity of the fusion protein at 3 mg/kg is temporary and reversible.
Example 15: In Vivo Anti-Tumor Activity of her-2 Antibody-TNFα Fusion Protein on her-2-Positive Mouse Colon Carcinoma CT26 in Mice
[0173] The Balb/c mice were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. Twenty one 6-7 weeks old male Balb/c mice were divided into 5 groups of 4 or 5 animals/group. The animals were subcutaneously grafted with CT26/Her-2 cells (1×10.sup.6 cells/animal) on back. When the tumor reached 100-400 mm.sup.3, the mice were injected intravenously with the antibody-TNFα fusion protein at different dosages: 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg and 3 mg/kg. The control group was injected with even volumes of PBS. The treatments were administrated three or four times a week for two weeks. Tumor volume was measured and animals weighed at each dosing. 6 days after the fourth dosing, the mice were sacrificed by cervical dislocation, and bodyweight, tumor volume and weight were recorded.
[0174] The in vivo anti-tumor effect of the Trastuzumab-TNFα fusion protein on the CT26/Her-2 tumor was further compared with that of the Trastuzumab+TNFα mixture. Twenty four 6-7 weeks old male Balb/c mice were divided into 3 groups of 8 animals/group. The animals were subcutaneously grafted with CT26/Her-2 cells (1×10.sup.6 cells/animal) on back. When the tumor reached 100-400 mm.sup.3, the three groups were injected intravenously with 0.2 ml PBS, Trastuzumab-TNFα fusion protein (3 mg/kg) and the Trastuzumab+TNFα mixture (3 mg/kg, the weight ratio of Trastuzumab to TNFα (dimeric) being 4:1). In the Trastuzumab+TNFα mixture, the molar ratio of Trastuzumab to TNFα was 1:2, the same as in the Trastuzumab-TNFα fusion protein. Two doses were given, with an interval of three weeks. Tumor volume and bodyweight were recorded at each dosing.
[0175] Results and Analysis
[0176] As shown in
[0177] As shown by these results, the Her-2 antibody-TNFα fusion protein can effectively inhibit growth of the CT26/Her-2 tumor and render a complete elimination of tumors in mice, producing an anti-tumor activity even at a dosage as low as 0.1 mg/kg.
[0178] The Trastuzumab-TNFα fusion protein was found far more efficacious than the Trastuzumab+TNFα mixture in inhibiting growth of the CT26/Her-2 tumor. The next day of second dosing (D13), in the group treated with 3 mg/kg of Trastuzumab-TNFα fusion protein, the average tumor volume was 33 mm.sup.3, while in the group of the Trastuzumab+TNFα mixture at a corresponding dosage, the average was 195 mm.sup.3, with inhibition rates being respectively 87% and 24% (relative to the average tumor volume in the PBS group being 257 mm.sup.3). The result was statistically significant, p<0.004 in both cases (
Description of Sequences
[0179]
TABLE-US-00003 SEQ ID NO: Name 1 Sequence encoding Trastuzumab's heavy chain-TNFα 2 Amino acid sequence of Trastuzumab's heavy chain-TNFα 3 Sequence encoding Trastuzumab's light chain 4 Amino acid sequence of Trastuzumab's light chain 5 Primer M13-F 6 Primer KDP004 7 Primer KDP045 8 Primer TNF-R 9 Primer M13-R 10 Primer KDP077 11 Primer KDP020 12 Primer BGH-R 13 Primer KDP092 14 Primer KDP093 15 Primer CMV-P 16 Primer KDP033 17 Primer KDP094 18 Primer KDP010 19 Sequence encoding Cetuximab's heavy chain-TNFα 20 Amino acid sequence of Cetuximab's heavy chain-TNFα 21 Sequence encoding Cetuximab's light chain 22 Amino acid sequence of Cetuximab's light chain